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. 2012 Nov 1;21(16):2987-3000.
doi: 10.1089/scd.2012.0099. Epub 2012 Aug 6.

FGF inhibition directs BMP4-mediated differentiation of human embryonic stem cells to syncytiotrophoblast

Smita Sudheer  1 , Raghu Bhushan, Beatrix Fauler, Hans Lehrach, James Adjaye
Affiliations

FGF inhibition directs BMP4-mediated differentiation of human embryonic stem cells to syncytiotrophoblast

Smita Sudheer et al. Stem Cells Dev. .

Abstract

Bone morphogenetic protein (BMP) signaling is known to support differentiation of human embryonic stem cells (hESCs) into mesoderm and extraembryonic lineages, whereas other signaling pathways can largely influence this lineage specification. Here, we set out to reinvestigate the influence of ACTIVIN/NODAL and fibroblast growth factor (FGF) pathways on the lineage choices made by hESCs during BMP4-driven differentiation. We show that BMP activation, coupled with inhibition of both ACTIVIN/NODAL and FGF signaling, induces differentiation of hESCs, specifically to βhCG hormone-secreting multinucleated syncytiotrophoblast and does not support induction of embryonic and extraembryonic lineages, extravillous trophoblast, and primitive endoderm. It has been previously reported that FGF2 can switch BMP4-induced hESC differentiation outcome to mesendoderm. Here, we show that FGF inhibition alone, or in combination with either ACTIVIN/NODAL inhibition or BMP activation, supports hESC differentiation to hCG-secreting syncytiotrophoblast. We show that the inhibition of the FGF pathway acts as a key in directing BMP4-mediated hESC differentiation to syncytiotrophoblast.

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Figures

FIG. 1.
FIG. 1.
Experimental design and BMP4-induced differentiation are accelerated and accentuated with additional inhibition of ACTIVIN/NODAL and FGF signaling. (A) Details of treatments [BMP4 (B): 10 ng/mL; SB431542 (SB): 20 μM; SU5402 (SU): 20 μM; FGF2 (F): 20 ng/mL] [Hour (H); Day (D)]. (B) Dendrogram based on Pearson's correlation coefficient between samples (5 days). (C) Principal Component Analysis on the regulated genes (2-fold), with respect to their expression in hESCs in at least one of the indicated (5 days). (D) Number of significantly regulated genes after 5 days of the indicated treatments, with respect to hESCs. Blue, downregulated; red, upregulated. (E) Western blot analysis for the indicated proteins and samples. (F) Heat map depicting the regulation of genes with respect to hESCs in the indicated samples. (G) Percentage overlaps of regulated genes (2-fold) in each sample (5 days) with respect to hESCs, with placental-regulated genes. BMP, bone morphogenetic protein; hESCs, human embryonic stem cells. Color images available online at www.liebertpub.com/scd
FIG. 2.
FIG. 2.
FGF and ACTIVIN/NODAL inhibition prevents BMP4-driven differentiation into mesendoderm and PE and specifically supports trophoblast induction. (A) Real-time PCR based analysis of T expression in the indicated samples. (B) Analysis of of EOMES expression in the indicated samples. (C) Analysis of CDH1, SNAI2 and VIM expression in the indicated samples (5 days). (D) Analysis of GATA6, SOX7, SOX17, and CER1 expression in the indicated samples. (E) Analysis of the indicated genes in B/SB/SU-treated samples (5 days). (F) Heat map depicting the regulation of genes with respect to hESCs in the indicated samples. PE, primitive endoderm. Color images available online at www.liebertpub.com/scd
FIG. 3.
FIG. 3.
B/SB/SU treatment transiently upregulates CDX2 and induces differentiation to βhCG-secreting syncytiotrophoblast and does not support extravillous trophoblast formation. (A) ELISA-mediated estimation of hCG concentration in growth media in the indicated samples. (B) Representative immunostain for βhCG expression in B/SB/SU-treated cells. (C) Representative immunostain for KRT7 expression in B/SB/SU-treated cells. (D) Analysis of OCT4, NANOG, and CGB expression in the indicated samples. (E) Western blot analysis of HLA-G expression in the indicated samples. (F) Representative immunostains for E-Cadherin (CDH1) and Vimentin (VIM) expression in B/SB/SU-treated cells. (G) Analysis of OCT4, SOX2, NANOG, and CGB, during B/SB/SU treatment. (H) Analysis of CDX2 expression in the indicated samples. (I) Western blot analysis of CDX2 expression in the indicated samples (5 days). Color images available online at www.liebertpub.com/scd
FIG. 4.
FIG. 4.
B/SB/SU treatment induces the expression of fusogens and differentiation to multinucleated syncytiotrophoblast. (A) Heat map and expression profile of coregulated and induced genes in comparison to hESCs during the B/SB/SU treatment. (B) Real-time PCR-based expression analysis of the indicated genes during B/SB/SU treatment. (C) Representative immunostain for E- Cadherin (CDH1) expression in the indicated samples. (D) Representative immunostain for CDH1 in the B/SB/SU-treated samples (5 days), exhibiting multinucleated regions in the colony edges and fusion indices of 3 different experiments. CDH1, green; DAPI, blue. (E) Representative immunostain for ß-Actin (ACTB) in the B/SB/SU-treated samples (5 days), exhibiting multinucleated regions in the colony edges. ACTB: yellow; DAPI: blue. (F) Representative electron microscopy images depicting multinucleated cells in B/SB/SU-treated samples (5 days). Scale bar: 5 μm. (G) Mitotic index for the indicated samples. (H) Representative immunostains for βhCG (green) and SYNCYTIN1 (red) expression in B/SB/SU-treated cells. DAPI, diamidino-2-phenylindoldihydrochloride. Color images available online at www.liebertpub.com/scd
FIG. 5.
FIG. 5.
Autocrine signaling leads to the abrogation of BMP signaling and WNT activation in B/SB/SU-treated cells (5 days). (A) Western blot analysis for the indicated proteins and samples. The 3-day (3D) samples were not considered for analysis due to unequal loading. (B) Expression analysis of GREM2 in the indicated samples. (C) Expression analysis of CHRD, FST, and GREM2 in the indicated samples. (D) Western blot analysis for the indicated proteins in B/SB/SU-treated samples. (E) Expression analysis of DKK1 in the indicated samples. GREM2, Gremlin2; CHRD, Chordin; DKK1, dickkopf homolog 1. Color images available online at www.liebertpub.com/scd
FIG. 6.
FIG. 6.
FGF inhibition in hESC supports differentiation to hCG-secreting syncytiotrophoblast. (A) ELISA-based estimation of hCG concentration in the growth medium after the indicated treatments. (B) Expression analysis of indicated genes in SU-treated samples (5 days). (C) Principal Component Analysis of regulated genes (2-fold) in the indicated samples. (D) Expression of CDX2, βhCG-encoding genes (CGB, CGB1, and CGB5), and HERV-FRD in the indicated samples, in comparison to hESCs (microarray data). (E) Western blot analysis for the expression of the indicated proteins and samples. (F) Representative immunostain for E-Cadherin (CDH1) expression in SU-treated cells. (G) Representative immunostain for SYNCYTIN1 (ERVWE1) and βhCG expression in SU-treated cells. (H) Representative immunostain for KRT7 expression in SU-treated cells. (I) Venn diagrams representing the overlap of the up- or downregulated genes between the indicated samples (5 days), in comparison to hESCs. (J) Heat map depicting the regulation of genes with respect to hESCs in the indicated samples. Color images available online at www.liebertpub.com/scd
FIG. 7.
FIG. 7.
A model summarizing the outcome of B/SB/SU treatment of hESCs that leads to syncytiotrophoblast induction (“+” and “−” represent activation and inhibition, respectively). FGF inhibition (SU) supports syncytiotrophoblast induction, but to a lesser extent compared to B/SB/SU treatment. The pathway–lineage map, prepared on the basis of our findings and that of other laboratories, represents the consolidated outcome of BMP4 activation and inhibition of both ACTIVIN/NODAL and FGF pathways (B/SB/SU). Dotted lines: the reason for the inhibition of PE induction from hESCs upon B/SB/SU treatment could be a combinatorial effect of inhibition of both ACTIVIN/NODAL and FGF, but this requires further investigation. Color images available online at www.liebertpub.com/scd
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